ABSTRACT HIV-1-associated inflammation and chronic immune activation persist despite suppressive antiretroviral therapy and are strongly linked to the development of comorbidities and increased mortality. The gastrointestinal (GI) tract is likely a major source of chronic inflammation, as early HIV-1 replication and CD4 T cell depletion in the gut results in mucosal inflammation and barrier dysfunction, leading to the translocation of enteric microbes/microbial products into the lamina propria and the systemic circulation. To gain insights into the molecular processes that drive gut-focused chronic immune activation, we profiled the transcriptome of gut CD4+CD8?- T cells exposed to gram-negative enteric bacteria and then infected with HIV-1 ex vivo. Multiple granzyme genes were upregulated following microbe exposure that was further enhanced with HIV-1 infection, suggesting the synergistic induction of CD4 cytotoxic T lymphocyte (CTL) activity. In pilot ex vivo studies, the human gut CD4 CTL phenotype was associated with granzyme (GZ) B expression, TCR signaling, Th1/17 polyfunctionality, reactivity to commensal bacteria and subsets expressing GZA, perforin and/or CD107a. The CD4 CTL phenotype was expressed in CD4 T cells from the gut to a much greater extent than from peripheral blood and lymphoid tissue. Importantly, HIV-1 replicated to a greater extent in gut CD4 CTLs ex vivo. Here, we hypothesize that gut CD4 CTLs are uniquely primed to respond to enteric microbes, are highly susceptible to HIV-1 mediated killing, and play critical roles in mucosal HIV-1 pathogenesis via cytolytic and pro-inflammatory mechanisms. To gain insights on the role of gut CD4 CTLs in HIV-1 pathogenesis, we propose 3 aims. In Aim 1, we will determine if microbiome species and ligands induce gut CD4 CTLs in an MHC-II-dependent manner, obtain insights into gut CD4 CTL origin and function by single-cell transcriptomics, and investigate the stability and fate of these cells ex vivo. In Aim 2, we will determine how HIV-1 infection further augments GZB production in gut CD4 T cells and using our ex vivo lamina propria aggregate culture (LPAC) model, determine if gut CD4 CTLs exacerbate direct and bystander HIV-1-mediated CD4 T cell death via cytolytic mechanisms. In Aim 3, we will evaluate the triggers for GZ secretion and dissect the non-cytolytic, pro-inflammatory properties of GZs secreted by microbe-exposed gut CD4 T cells ex vivo. Importantly, we will explore the in vivo relationship between CD4 CTL frequencies and markers of mucosal and systemic inflammation in archived plasma and gut biopsies from well-characterized cohorts of both untreated and treated persons with chronic HIV-1 infection. Altogether, these studies should provide critical information on this striking gut immune cell subpopulation and its contribution to mucosal inflammation and disease.